We used a fluorescence microscope described in detail in our previous studies (Oyama et al., 2012 (link); Shintani et al., 2015 (link)). The optical setup was built around an inverted microscope IX70 (Olympus) with an objective lens (PlanApo N 60×/1.45 Oil; Olympus). Namely, a stable light source, Light Engine (with wavelengths of 549/15 nm and 377/50 nm; Lumencor), was used for excitation of actin filaments stained with rhodamine–phalloidin and a thermosensor sheet. A dichroic mirror (FF562-Di02; Semrock) and an emission filter (BA580IF; Olympus) were mounted. The solution was directly heated by focusing the IR-laser beam (λ = 1,455 nm, for 2 s; KPS-STD-BT-RFL-1455-02-CO; Keopsys) under the microscope. The laser power was 33 mW, which was measured at the top of the objective lens by using a thermal disk sensor and a power meter (LM-3 and FieldMaster; Coherent). The ON/OFF of heating was regulated by a shutter system (SSH-C4B; SIGMAKOKI) placed in the light path of the IR laser beam. Fluorescence images were recorded using an electron-multiplying charge-coupled device camera (iXon EM+ 897; Andor Technology) at 33 frames per second and stored in a Windows PC via ANDOR IQ software (Andor Technology). The present experimental setup is illustrated in Fig. 1 A .
Ixonem 897
Manufactured by Oxford Instruments
Sourced in Japan, United Kingdom
The IXonEM+ 897 is a high-performance, electron-multiplying CCD (EMCCD) camera designed for low-light imaging and spectroscopy applications. It features a back-illuminated, 1024 x 1024 pixel sensor with an effective pixel size of 13 μm. The camera is capable of achieving high quantum efficiency, low readout noise, and high EM gain, making it suitable for a wide range of scientific research and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Andor iq software, by Oxford Instruments (2 mentions)
Ff562 di02, by IDEX Corporation (2 mentions)
Phoxx 642, by Toptica (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Coolsnap hq, by Teledyne (1 mentions)
Dmi6000, by Leica (1 mentions)
Pluronic acid, by Thermo Fisher Scientific (1 mentions)
Ix70 microscope, by Olympus (1 mentions)
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lambda 10 3 filter wheel, by Sutter Instruments (1 mentions)
600 677 nm brightline quad band band pass filter, by IDEX Corporation (1 mentions)
Uapon 100x 1.49na, by Olympus (1 mentions)
Diode laser, by Toptica (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
Cylindrical lens, by Thorlabs (1 mentions)
488 nm laser, by Toptica (1 mentions)
9 protocols using ixonem 897
1
Fluorescence Microscopy for Thermosensor Imaging
2
Visualizing Alae Defects in Live C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Live young adult animals were immobilized with 0.1% tricaine/0.01% tetramisole and mounted on 2% agarose pads at 20°C. Alae defects phenotypes were observed by differential interference contrast using a Leica DMRXA2 6000 equipped with a Coolsnap HQ (Roper Scientific) camera with a 100× objective (oil; NA 1.4). To follow the localization of fluorescent proteins, Z-stacks (0.3-µm spacing) were acquired using a DMI6000 (Leica) spinning disk (Yokogawa CSU22 with an Andor iXonEM+ 897 camera) with a 63× objective (oil; NA 1.4). Leica Type F immersion medium was used. Young adults were observed (right after alae formation and before formation of the first embryos). Identical settings were used for control and mutant animals. Image analysis was performed using ImageJ software (National Institutes of Health). Colocalization (Fig. 1 , Fig. 2 , and Fig. 4 ) was quantified using a semiautomated method (comparison of local intensity maxima obtained in each channel) and a manual method based on scan line analysis (comparison of fluorescence intensity profile along a line crossing a fluorescent punctum in a given channel with the profile obtained from the same line in the other channel), which was previously described (Hyenne et al., 2012 (link)).
3
Ca2+ Imaging of Gallbladder Whole Mounts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Imaging of Ca2+ events in GB whole mount preparations was performed as previously described [2] (link). Briefly, the GB was removed and opened from fundus to the cystic duct in ice-cold modified Krebs solution. The full-thickness whole mounts were stretched open and pinned serosa side up between two pieces of Sylgard connected by metal pins. Tissue was then loaded for 1 h at room temperature in a HEPES buffer containing 10 μM fluo-4AM (F14201,Thermo-Fisher Scientific, Waltham, MA) and 2.5 μg/ml pluronic acid (#24040032, Thermo-Fisher Scientific). Following washing and incubation for at least 30 min at room temperature to allow de-esterification, the preparations were placed in a Ca2+ imaging chamber and superfused with aerated physiological saline solution at 35–37 °C. After a 15–20 min equilibration, Ca2+ events were visualized using an Andor iXonEM + 897 back-illuminated EMCCD camera attached to an inverted fluorescent Olympus IX70 microscope equipped with a 40X objective. Movies were acquired over periods of 20–30 s (30 frames per second) and analyzed using SparkAN, a custom software written at the University of Vermont (A. D. Bonev).
4
Drosophila Lipid Droplet Protein Imaging
5
Yeast Fluorescence Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For fluorescence microscopy, yeast cells were grown to OD = 0.6 in synthetic medium at 30°C unless otherwise indicated. Cells were mounted in synthetic media onto coverslips previously coated with concanavalin A and imaged with a spinning-disk confocal microscope (TiLL iMIC CSU22; Andor, Northern Ireland) using a back-illuminated EM charge-coupled device camera (iXonEM 897; Andor) and a 100 × 1.4 NA oil immersion objective (Olympus, Japan). 16-bit images were collected using Image iQ (version 1.9; Andor). Images were filtered with a smoothening filter averaging 2 pixels, converted to 8-bit images, and cropped using ImageJ software (http://rsbweb.nih.gov/ij/ ).
6
Inverted Microscopy for Fluorescence Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The microscope was similar to the one that was previously used14 (link). An IX70 inverted microscope with a PlanApo N 60×/1.45 oil objective lens (Olympus, Tokyo, Japan) was mounted on an optical bench. A mercury lamp and Lambda 10-3 filter wheel (Sutter Instrument, CA, USA) were set outside the bench and connected to the microscope via a liquid light guide. To prevent excessive bleaching of the fluorescence signal, neutral density filters were placed in front of a liquid light guide. Fluorescence images were recorded with BP360-370, DM505, and BA515IF (respectively, excitation filter, dichroic mirror, and emission filter) for thermometer sheet; BP470-490, DM505, and BA515IF (a green channel) for Ca2+ dynamics; BP520-550, FF562-Di02, and BA580IF (a red channel) for injection marker, respectively (FF562-Di02, Semrock, NY, USA; others, Olympus). For bright field and fluorescence imaging, an iXon EM+ 897 electron multiplying charge-coupled device camera shooting an average of 9.9 frames/s and Andor iQ software (Andor Technology, Antrim, UK) were used along with an FF01-790 short-pass filter (Semrock) placed in front of the camera.
7
Dual-color Imaging of Platelet Ultrastructure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were acquired using a modified Olympus IX81 inverted epifluorescence microscope with an oil-immersion objective (UApo N 100x/1.49 NA, Olympus, Vienna, Austria). The sample was positioned with nanometer precision on a XYZ piezo stage (P-733.3DD, Physical Instruments) on top of a mechanical stage with a range of 1 × 1 cm adjusted by precision screws (TAO, JPK Instruments, Berlin, Germany). A tube-lens with an additional magnification of 1.6 was used to achieve a final imaging magnification of 160 (corresponding to a pixel size of 100 nm). Platelets were illuminated with a 642 nm laser light from a diode laser (Omicron-laserage Laserprodukte GmbH, Phoxx 642, Rodgau-Dudenhofen, Germany), a 488 nm laser light from a solid-state laser (diode-pumped, Toptica Photonics, Graefelfing, Germany), and a 405 nm laser light from a diode laser (Insaneware, Gladbeck, Germany). The signal was detected using an Andor iXonEM+ 897 (back-illuminated) EMCCD camera (16 μm pixel size). The following filter sets were used: dichroic filter (ZT405/488/561/640rpc, Chroma, Olching, Germany), emission filter (446/523/600/677 nm BrightLine quad-band band-pass filter, Semrock, Rochester, NY, USA), and an additional emission filter (HQ 700/75 M, NC209774, Chroma Technology GmbH, Olching, Germany).
8
Single-Molecule Fluorescence Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The images were taken on a modified Olympus IX81 inverted microscope. The samples were illuminated through an Olympus UApo N 100×/1.49 NA oil objective with diode laser at 532 nm (Cobolt Calypso 100 TM). The signal acquisition was carried out on an Andor iXonEM + 897 (back illuminated) EMCCD (160 nm pixel size). The experiments were performed using excitation powers of 0.025 kW/cm2 at 532 nm. The samples were illuminated for 5 ms with 35 ms delay time. The illumination protocols were timed with a custom made LabView® based control software. Filter: Overlay (642/532), Dichroic filter: Cy3/Cy5, Emission-filter: Cy3/Cy5 + Bandpass 595/50 (Chroma). The cells were imaged in two illumination configurations, the widefield and highly inclined and laminated optical light sheet (HILO) illumination. The light sheet illumination reduces background fluorescence within the cell, originating from scattered light or other fluorescent molecules [27 (link)].
9
High-resolution 3D Fluorescence Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were acquired using a modified Olympus
IX81 inverted epifluorescent
microscope with an oil-immersion objective (PlanApo N, 60×, NA
1.42, Olympus, Vienna, Austria). Samples were mounted on a XYZ piezo
stage (PI Mars; P-562, Physical Instruments) which has nanometer accuracy,
combined with a coarse mechanical stage with a travel range of 1 cm
× 1 cm (Hybrid, JPK Instruments, Berlin, Germany). A tube lens
with an additional magnification of 1.6 was used to achieve a final
imaging magnification of 96 (corresponding to a pixel size of 167
nm). ECs were illuminated with a 642 nm laser diode (Omicron-laserage
Laserprodukte GmbH, Phoxx 642, Rodgau-Dudenhofen, Germany) and a 488
nm laser (Toptica Photonics, Germany). Signals were collected using
an Andor iXonEM+ 897 (back-illuminated) EMCCD camera (16 μm
pixel size). The following filter sets were used: dichroic filter
(ZT405/488/561/640rpc, Chroma, Germany), emission filter (446/523/600/677
nm BrightLine quad-band band-pass filter, Semrock, Rochester, NY,
USA), and additional emission filters: ET 700/75 M, Chroma Technology
GmbH, Olching, Germany; ET 525/50 M, Chroma Technology GmbH, Olching,
Germany. For 3D measurements, a cylindrical lens (f = 500 mm; Thorlabs, Newton, USA) was placed into the detection path
of the microscope.
IX81 inverted epifluorescent
microscope with an oil-immersion objective (PlanApo N, 60×, NA
1.42, Olympus, Vienna, Austria). Samples were mounted on a XYZ piezo
stage (PI Mars; P-562, Physical Instruments) which has nanometer accuracy,
combined with a coarse mechanical stage with a travel range of 1 cm
× 1 cm (Hybrid, JPK Instruments, Berlin, Germany). A tube lens
with an additional magnification of 1.6 was used to achieve a final
imaging magnification of 96 (corresponding to a pixel size of 167
nm). ECs were illuminated with a 642 nm laser diode (Omicron-laserage
Laserprodukte GmbH, Phoxx 642, Rodgau-Dudenhofen, Germany) and a 488
nm laser (Toptica Photonics, Germany). Signals were collected using
an Andor iXonEM+ 897 (back-illuminated) EMCCD camera (16 μm
pixel size). The following filter sets were used: dichroic filter
(ZT405/488/561/640rpc, Chroma, Germany), emission filter (446/523/600/677
nm BrightLine quad-band band-pass filter, Semrock, Rochester, NY,
USA), and additional emission filters: ET 700/75 M, Chroma Technology
GmbH, Olching, Germany; ET 525/50 M, Chroma Technology GmbH, Olching,
Germany. For 3D measurements, a cylindrical lens (f = 500 mm; Thorlabs, Newton, USA) was placed into the detection path
of the microscope.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!